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Optimization of isolation and further characterization of umbilical cord blood‐derived very small embryonic/ epiblast‐like stem cells (VSELs)
Author(s) -
ZubaSurma Ewa K.,
Klich Iza,
Greco Nick,
Laughlin Mary J.,
Ratajczak Janina,
Ratajczak Mariusz Z.
Publication year - 2010
Publication title -
european journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0902-4441
DOI - 10.1111/j.1600-0609.2009.01352.x
Subject(s) - embryonic stem cell , epiblast , ficoll , umbilical cord , cell sorting , stem cell , population , microbiology and biotechnology , andrology , chemistry , immunology , biology , flow cytometry , biochemistry , medicine , peripheral blood mononuclear cell , in vitro , gastrulation , environmental health , gene
Because of their small size and density, umbilical cord blood (UCB)‐derived very small embryonic/epiblast‐like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll‐Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three‐step isolation protocol that allows recovery of approximately 60% of the initial number of Lin − /CD45 − /CD133 + UCB‐VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133 + including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin − /CD45 − /CD133 + cells are sorted by fluorescence‐activated cell sorting. The whole isolation procedure takes approximately 2–3 h per UCB unit and isolated cells are highly enriched for an Oct‐4 + and SSEA‐4 + population of small Lin − /CD45 − /CD133 + cells.